Author Correction: Rapid and efficient induction of functional astrocytes from human pluripotent stem cells.

In the version of Supplementary Fig. 1 originally published with this paper, some images in panel e were accidental duplicates of images in panel b. This error has been corrected in the online integrated supplementary information and in the Supplementary Information PDF.

Rapid and efficient induction of functional astrocytes from human pluripotent stem cells.

The derivation of astrocytes from human pluripotent stem cells is currently slow and inefficient. We demonstrate that overexpression of the transcription factors SOX9 and NFIB in human pluripotent stem cells rapidly and efficiently yields homogeneous populations of induced astrocytes. In our study these cells exhibited molecular and functional properties resembling those of adult human astrocytes and were deemed suitable for disease modeling. Our method provides new possibilities for the study of human astrocytes in health and disease.

Monocyte-Derived Macrophages Contribute to Spontaneous Long-Term Functional Recovery after Stroke in Mice.

Stroke is a leading cause of disability and currently lacks effective therapy enabling long-term functional recovery. Ischemic brain injury causes local inflammation, which involves both activated resident microglia and infiltrating immune cells, including monocytes. Monocyte-derived macrophages (MDMs) exhibit a high degree of functional plasticity. Here, we determined the role of MDMs in long-term spontaneous functional recovery after middle cerebral artery occlusion in mice. Analyses by flow cytometry and immunocytochemistry revealed that monocytes home to the stroke-injured hemisphere., and that infiltration peaks 3 d after stroke. At day 7, half of the infiltrating MDMs exhibited a bias toward a proinflammatory phenotype and the other half toward an anti-inflammatory phenotype, but during the subsequent 2 weeks, MDMs with an anti-inflammatory phenotype dominated. Blocking monocyte recruitment using the anti-CCR2 antibody MC-21 during the first week after stroke abolished long-term behavioral recovery, as determined in corridor and staircase tests, and drastically decreased tissue expression of anti-inflammatory genes, including TGFß, CD163, and Ym1. Our results show that spontaneously recruited monocytes to the injured brain early after the insult contribute to long-term functional recovery after stroke. SIGNIFICANCE STATEMENT: For decades, any involvement of circulating immune cells in CNS repair was completely denied. Only over the past few years has involvement of monocyte-derived macrophages (MDMs) in CNS repair received appreciation. We show here, for the first time, that MDMs recruited to the injured brain early after ischemic stroke contribute to long-term spontaneous functional recovery through inflammation-resolving activity. Our data raise the possibility that inadequate recruitment of MDMs to the brain after stroke underlies the incomplete functional recovery seen in patients and that boosting homing of MDMs with an anti-inflammatory bias to the injured brain tissue may be a new therapeutic approach to promote long-term improvement after stroke.

Transcription factor programming of human ES cells generates functional neurons expressing both upper and deep layer cortical markers.

Human neurodegenerative disorders affect specific types of cortical neurons. Efficient protocols for the generation of such neurons for cell replacement, disease modeling and drug screening are highly warranted. Current methods for the production of cortical neurons from human embryonic stem (ES) cells are often time-consuming and inefficient, and the functional properties of the generated cells have been incompletely characterized. Here we have used transcription factor (TF) programming with the aim to induce rapid differentiation of human ES cells to layer-specific cortical neurons (hES-iNs). Three different combinations of TFs, NEUROGENIN 2 (NGN2) only, NGN2 plus Forebrain Embryonic Zinc Finger-Like Protein 2 (FEZF2), and NGN2 plus Special AT-Rich Sequence-Binding Protein 2 (SATB2), were delivered to human ES cells by lentiviral vectors. We observed only subtle differences between the TF combinations, which all gave rise to the formation of pyramidal-shaped cells, morphologically resembling adult human cortical neurons expressing cortical projection neuron (PN) markers and with mature electrophysiological properties. Using ex vivo transplantation to human organotypic cultures, we found that the hES-iNs could integrate into adult human cortical networks. We obtained no evidence that the hES-iNs had acquired a distinct cortical layer phenotype. Instead, our single-cell data showed that the hES-iNs, similar to fetal human cortical neurons, expressed both upper and deep layer cortical neuronal markers. Taken together, our findings provide evidence that TF programming can direct human ES cells towards cortical neurons but that the generated cells are transcriptionally profiled to generate both upper and deep layer cortical neurons. Therefore, most likely additional cues will be needed if these cells should adopt a specific cortical layer and area identity.

Direct conversion of human fibroblasts to functional excitatory cortical neurons integrating into human neural networks.

BACKGROUND: Human fibroblasts can be directly converted to several subtypes of neurons, but cortical projection neurons have not been generated. METHODS: Here we screened for transcription factor combinations that could potentially convert human fibroblasts to functional excitatory cortical neurons. The induced cortical (iCtx) cells were analyzed for cortical neuronal identity using immunocytochemistry, single-cell quantitative polymerase chain reaction (qPCR), electrophysiology, and their ability to integrate into human neural networks in vitro and ex vivo using electrophysiology and rabies virus tracing. RESULTS: We show that a combination of three transcription factors, BRN2, MYT1L, and FEZF2, have the ability to directly convert human fibroblasts to functional excitatory cortical neurons. The conversion efficiency was increased to about 16% by treatment with small molecules and microRNAs. The iCtx cells exhibited electrophysiological properties of functional neurons, had pyramidal-like cell morphology, and expressed key cortical projection neuronal markers. Single-cell analysis of iCtx cells revealed a complex gene expression profile, a subpopulation of them displaying a molecular signature closely resembling that of human fetal primary cortical neurons. The iCtx cells received synaptic inputs from co-cultured human fetal primary cortical neurons, contained spines, and expressed the postsynaptic excitatory scaffold protein PSD95. When transplanted ex vivo to organotypic cultures of adult human cerebral cortex, the iCtx cells exhibited morphological and electrophysiological properties of mature neurons, integrated structurally into the cortical tissue, and received synaptic inputs from adult human neurons. CONCLUSIONS: Our findings indicate that functional excitatory cortical neurons, generated here for the first time by direct conversion of human somatic cells, have the capacity for synaptic integration into adult human cortex.